Candidate: Majoring as analytical chemist during the time in Belgrade, Serbia, Dr. Gligorijevic has become an expert in optics and live cell microscopy over the course of her graduate training at Georgetown University and developed several novel microscopy-based technologies. During the postdoctoral period, Dr. Gligorijevic has focused on studying tumor cell intravasation in breast tumors, in living animals. To do so, she introduced several advancements into in vivo multiphoton microscopy. Dr. Gligorijevic is funded by DOD and Charles Revson Fellowship and has received several honors for her research. Her work to date resulted in one patent, ten peer-reviewed and ten other publications. Recent research has shown that different components of tumor microenvironment have an essential role in tumor progression. In order to decipher the network of interactions which shape the tumor environment and determine tumor cell behavior, it is ideal to apply systems biology approaches combined with in vivo microscopy, as cell cultures are limited in their complexity, mouse models are time-consuming and expensive and in silico models need optimization based on experiments. During the mentored phase of the Award, Dr. Gligorijevic will incorporate the relevant principles of advanced statistics and mathematical modeling of dynamical systems into her background of tumor biology and microscopy. She will take coursework offered at Einstein and meet regularly with her sponsor and co-sponsor. This training will make it possible to build a laboratory which investigates mechanisms of metastasis using the integrative, systems microscopy approach and utilizing information from molecular to population levels. Environment: Sponsor of the PI, Dr. John Condeelis is a Co-Chair of Anatomy Department and Biophotonic Center at Einstein. His lab and the Center create a multidisciplinary environment focused on answering mechanisms of human diseases, such as cancer, through use of microscopy. The Center is well known for its shared imaging resources and Innovation Laboratory where new microscopy systems are custom-built to accommodate specific needs of different projects. Consulting member of Advisory Committee, Dr. Robert Singer, is an expert in combining experimental and theoretical biology and a renowned mentor. The co-sponsor, Dr. Aviv Bergman, is the Founding Chair of Systems Department at Einstein and teaches coursework which will be a part of the career development. Einstein is an institution which highly values collaboration and insists on career development of postdoctoral fellows, instructors and junior faculty. Research: While most research of tumor microenvironments focuses on isolating and understanding single parameters, an integrative, systems-level network of interactions among relevant biological players is missing. In primary tumors, there are numerous biomechanical signals able to direct tumor cell movement towards and into the blood vessels. Growth factors, secreted by host macrophages, fibroblasts and endothelial cells in the tumor microenvironment are the main chemoattractants but recent studies show that the extracellular matrix also plays an important role. In loose extracellular matrix, tumor cells can migrate by reorganizing their cytoskeleton, generating a protrusive force. In regions with stiff extracellular matrix, simple locomotion is not possible. Here, tumor cells become invasive and degrade extracellular matrix, mainly by matrix metalloproteases (MMPs). Tumor cell migration was well characterized in vivo, but the mechanism of the switch from locomotory to invasive state and assembly of invasive protrusions in vivo are unknown. To address the link between integrated microenvironment signaling and the tumor cell behavior, it is necessary to combine cell biology, advanced microscopy and systems biology. In preliminary experiments, tumor cells were recorded using time-resolved 3D multiphoton imaging in living mice and two different protrusion types were observed: a) locomotory protrusions which quickly lead to migration of the tumor cell and b) invasive protrusions, which are persistent and MMP-dependent. The goal of Aim 1 is to investigate the role of invasive protrusions in the tumor cell intravasation and metastasis. A combination of fluorescent reporters will be used for imaging of invasive protrusion assembly and investigation of consequent tumor cell fate. Aim 2 explores signals which drive tumor cells to form either locomotory or invasive protrusions or to switch between behaviors. In areas which contain either locomotory or invasive protrusions, tumor microenvironment parameters are recorded simultaneously (number of macrophages, collagen stiffness, blood vessel size etc.). Imaging analysis results in a data matrix, which is analyzed by a Support Vector Machine classification. Classification shows that tumor cells switch from locomotory to invasive states under very specific conditions. Proposed experiments test the hypothesis that tumor cell behavior can be changed by slightly modifying microenvironment parameters. Relevant microenvironment parameters are incorporated into an integrative mathematical model of the tumor cell switch from locomotory to invasive state, in Aim 3. Using the predictive power of the model, experiments were designed to inhibit the invasive state and subsequent metastasis. Experimental outcomes will be used to optimize and complexify the model. Results of this study will lead to better understanding of the interplay among microenvironment components during tumor progression and the results will be used to improve diagnosis and treatment of early metastasis.